Reproducibility of saliva progesterone measured by immunoassay compared to liquid chromatography mass spectrometry.

Immunoassay overestimates progesterone in blood, but no studies have tested whether this occurs in saliva. We measured progesterone in saliva using immunoassay and mass spectrometry. We tested the immunoassay for cross reactivity with dehydroepiandrosterone sulfate (DHEA-S) and 17α-hydroxyprogesterone (17α-OHP). Progesterone was significantly higher in immunoassay compared to mass spectrometry. Immunoassay progesterone levels increased in when incremental levels of 17α-OHP standard was added. This effect was not observed with the addition of DHEA-S. Research using salivary progesterone immunoassay techniques should be wary, particularly with individuals taking steroid supplementation or with high levels of progesterone metabolites.

Diosgenone synthesis, anti-malarial activity and QSAR of analogues of this natural product.

Solanum nudum Dunal steroids have been reported as being antimalarial compounds; however, their concentration in plants is low, meaning that the species could be threatened by over-harvesting for this purpose. Swern oxidation was used for hemisynthesis of diosgenone (one of the most active steroidal sapogenin diosgenin compounds). Eighteen structural analogues were prepared; three of them were found to be more active than diosgenone (IC50 27.9 µM vs. 10.1 µM, 2.9 µM and 11.3 µM). The presence of a 4-en-3-one grouping in the A-ring of the compounds seems to be indispensable for antiplasmodial activity; progesterone (having the same functional group in the steroid A-ring) has also displayed antiplasmodial activity. Quantitative correlations between molecular structure and bioactivity were thus explored in diosgenone and several derivatives using well-established 3D-QSAR techniques. The models showed that combining electrostatic (70%) and steric (30%) fields can explain most variance regarding compound activity. Malarial parasitemia in mice became reduced by oral administration of two diosgenone derivatives.

Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry.

Assessing the amount of bioavailable cortisol in saliva with immunoassays and thus sampling an endocrine marker of hypothalamus-pituitary-adrenal axis activity is of major interest in both research and clinical practice. However, absolute cortisol concentrations obtained with different immunoassays (IAs) are barely comparable precluding direct comparison between studies or individuals whenever cortisol analyses were not based on the same IA. The present technical report aims to solve this problem by evaluating the validity of, as well as agreement between the most commonly used immunoassays in psychoneuroendocrinological research (i.e., IBL, DRG, Salimetrics, DSL, and DELFIA) and a reference method (LC-MS/MS) in a sample of 195 saliva specimen covering the whole range of cortisol concentrations in adults. A structural equation modelling framework is applied to decompose systematic assay variance and estimate cortisol reference values, which are adjusted for measurement error and interference of salivary cortisone. Our findings reveal nonlinear relations between IAs and LC-MS/MS, which are discussed in terms of IA cross-reactivity with saliva matrix components. Finally guidelines for converting cortisol concentrations being obtained by these immunoassays into comparable reference values are proposed by providing conversion functions, a conversion table, and an online conversion tool.

[Effects of Yangling Zhongyu Decoction on the secretion of ovarian granule cells in polycystic ovarian syndrome rat model].

OBJECTIVE: To observe the effects of Yangjing Zhongyu Decoction (YJZYD) on the serum estradiol (E2), testosterone (T), 17-hydroxyprogesterone (17-OHP), ovarian follicle stimulating hormone receptor (FSHR), insulin-like growth factor I (IGF-1), steroid hormone acute regulator protein (StAR) mRNA expressions in female rats. METHODS: Fifty PCOS rats were equally divided into 5 groups, i. e., the control group (C, normal PNA rats), the model group (M), the low dose YJZYD group (Y1), the medium dose YJZYD group (Y2), and the high dose YJZYD group (Y3), 10 in each. The levels of serum hormones were detected using radioimmunoassay. The morphological changes of the ovary were observed using HE method. The expressions of FSHR, IGF-1, and StAR mRNA were detected using RT PCR. RESULTS: Compared with Group C, serum T and 17-OHP significantly increased (P < 0.01), E2 significantly decreased (P < 0.01), the expressions of FSHR, IGF-1, and StAR mRNA significantly decreased in Group M (P < 0.01). Compared with Group M, the serum T level significantly decreased (P < 0.01), 17-OHP decreased (P < 0.05), and E2 significantly increased in Group Y3 (P < 0.01). The expressions of FSHR, IGF-1, and StAR mRNA increased in Group Y1, Y2, and Y3. The increase was most obvious in Group Y3 (P < 0.01). CONCLUSIONS: YJZYD could lower the hyperandrogenemia of PCOS rats. It also could increase the ovarian expressions of FSHR, IGF-1, and StAR mRNA, improve the ovarian functions, and promote the follicular development.

Evaluation of serum concentrations of cortisol and sex hormones of adrenal gland origin after stimulation with two synthetic ACTH preparations in clinically normal dogs.

OBJECTIVE: To compare the adrenocortical response of healthy dogs to a commonly used dose of a nonadsorbed tetracosactide product (tetracosactide) with responses to 2 doses of a depot formulation of tetracosactide (depot tetracosactide). ANIMALS: 14 dogs. PROCEDURES: Dogs were randomly assigned to receive tetracosactide (5 mg/kg, IV) or depot tetracosactide (250 µg, IM, or 5 µg/kg, IM). Dogs received each treatment once with a 2-week interval between treatments. Blood samples were assayed for cortisol, progesterone, 17-hydroxyprogesterone, androstenedione, and estradiol concentrations. RESULTS: Serum cortisol concentrations were significantly higher than the preadministration (baseline) concentrations for all treatments 60 minutes after administration of ACTH. Peak cortisol concentration was detected 180 minutes after IM administration of 250 µg of the depot tetracosactide. Serum concentrations of progesterone, 17-hydroxyprogesterone, and androstenedione did not differ significantly from baseline concentrations after stimulation with the 5 µg/kg dose of depot tetracosactide. Adrenal gland progesterone response was significantly higher than baseline concentrations at 60 minutes after administration of the 250-µg dose of depot tetracosactide, and the 17-hydroxyprogesterone and androstenedione responses were significantly higher than baseline concentrations at 120 minutes. Compared with the response to tetracosactide, adrenocortical response was higher and more sustained following administration of the depot tetracosactide, except for androstenedione concentration, which had a nonsignificant response. CONCLUSIONS AND CLINICAL RELEVANCE: Except for androstenedione concentrations, a high dose of the depot tetracosactide (250 µg, IM) induced an adrenocortical response similar to that after administration of tetracosactide. Thus, depot tetracosactide may represent an alternative to the nonadsorbed tetracosactide product.

Molecular testing in congenital adrenal hyperplasia due to 21α-hydroxylase deficiency in the era of newborn screening.

Newborn screening (NBS) identifies the majority of classical [salt-wasting (SW) and simple-virilizing (SV)] cases of congenital adrenal hyperplasia (CAH) due to 21α-hydroxylase (21α-OHase) during the first days of life. Diagnosis of classical CAH is confirmed by follow-up serum 17-hydroxyprogesterone and/or the adrenocorticotropin stimulation test; however, neither test definitively distinguishes between the classical subtypes. After confirmation, all newborns are started on hydrocortisone (glucocorticoid) and fludrocortisone (mineralocorticoid) treatment. While initiating fludrocortisone treatment in classical CAH patients, independent of subtype and before SW signs or symptoms occur, prevents a life-threatening SW crisis, it may later complicate distinguishing between the classical subtypes. Genotype-phenotype correlations in 21α-OHase deficiency are excellent; however, molecular testing is not a regular part of the diagnostic workup. Molecular testing on 39 patients (25 identified by NBS) with an already established diagnosis of CAH identified 11 SW patients (8 identified by NBS) whose mutations suggested further biochemical and clinical reassessment of their subtype. Overall, SW accounted for 57.6% of our classical CAH patients, below the generally accepted figure that >75% of classical CAH are comprised of the SW form. In the era of NBS, molecular testing is a valuable supplemental tool identifying patients who may benefit from reassessment of their salt-retaining ability.

Androgens concentrations and second-to fourth-digit ratio (2D:4D) in girls with congenital adrenal hyperplasia (21-hydroxylase deficiency).

OBJECTIVES: Excessive hyperandrogenism, though proper hydrocortisone supplementation is a frequent clinical problem in girls with congenital adrenal hyperplasia (CAH). This may result from autonomic regulation of androgen production established in prenatal life. It has been suggested that the length of the second finger relative to the length of the fourth finger (2D;4D ratio) is negatively related to prenatal testosterone concentration. DESIGN AND SETTING: The retrospective study aimed to establish the relationship between the level of androgenization in utero determined using 2D:4D ratio and serum androgen concentrations in treated girls with CAH (21-OH deficiency) has been performed on 19 girls with CAH (21-OH deficiency) at the age of 3.7-19 years (mean 13.8 ± 4.07 years). All subjects were adequately treated with hydrocortisone (10-19 mg/m2; mean 13.81 ± 4.07 mg/m2). Anthropometric measurements of digits length were performed in all girls on X-rays obtained for bone age estimation. Apart from it, serum androgens concentrations (testosterone, androstenedione, s-DHEA) and 17-OH-progesterone (17-OHP) were assayed. RESULTS: Mean androgens serum concentrations in examined group were: testosterone 150.21 ± 155.44 ng/ml; androstenedione 4.15 ± 5.32 ng/ml, s-DHEA 70.39 ± 85.52 µg/dl. Mean 2D:4D ratio was 0.96 ± 0.04. Analysis of correlation showed positive linear correlations between testosterone, s-DHEA and 2D:4D ratio (r=0.53, p=0.023 and r=0.53; p=0.019, respectively). CONCLUSIONS: 2D:4D ratio parameter may be a simple test in indentification of female CAH patients prone to excessive androgen secretion despite proper treatment. The autonomization of adrenal androgens production in foetal life may cause its elevated levels in female patients with CAH although treated adequately.

Effects of Yangling Zhongyu Decoction on the secretion of ovarian granule cells in polycystic ovarian syndrome rat model / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effects of Yangjing Zhongyu Decoction (YJZYD) on the serum estradiol (E2), testosterone (T), 17-hydroxyprogesterone (17-OHP), ovarian follicle stimulating hormone receptor (FSHR), insulin-like growth factor I (IGF-1), steroid hormone acute regulator protein (StAR) mRNA expressions in female rats.</p><p><b>METHODS</b>Fifty PCOS rats were equally divided into 5 groups, i. e., the control group (C, normal PNA rats), the model group (M), the low dose YJZYD group (Y1), the medium dose YJZYD group (Y2), and the high dose YJZYD group (Y3), 10 in each. The levels of serum hormones were detected using radioimmunoassay. The morphological changes of the ovary were observed using HE method. The expressions of FSHR, IGF-1, and StAR mRNA were detected using RT PCR.</p><p><b>RESULTS</b>Compared with Group C, serum T and 17-OHP significantly increased (P < 0.01), E2 significantly decreased (P < 0.01), the expressions of FSHR, IGF-1, and StAR mRNA significantly decreased in Group M (P < 0.01). Compared with Group M, the serum T level significantly decreased (P < 0.01), 17-OHP decreased (P < 0.05), and E2 significantly increased in Group Y3 (P < 0.01). The expressions of FSHR, IGF-1, and StAR mRNA increased in Group Y1, Y2, and Y3. The increase was most obvious in Group Y3 (P < 0.01).</p><p><b>CONCLUSIONS</b>YJZYD could lower the hyperandrogenemia of PCOS rats. It also could increase the ovarian expressions of FSHR, IGF-1, and StAR mRNA, improve the ovarian functions, and promote the follicular development.</p>

Cryptotanshinone reverses reproductive and metabolic disturbances in prenatally androgenized rats via regulation of ovarian signaling mechanisms and androgen synthesis.

This trial explores 1) prenatally androgenized (PNA) rats as a model of polycystic ovary syndrome (PCOS) and 2) reproductive and metabolic effects of cryptotanshinone in PNA ovaries. On days 16-18 of pregnancy, 10 rats were injected with testosterone propionate (PNA mothers) and 10 with sesame oil (control mothers). At age 3 mo, 12 female offspring from each group were randomly assigned to receive saline and 12 cryptotanshinone treatment during 2 wk. Before treatment, compared with the 24 controls, the 24 PNA rats had 1) disrupted estrous cycles, 2) higher 17-hydroxyprogesterone (P = 0.030), androstenedione (P = 0.016), testosterone and insulin (P values = 0.000), and glucose (P = 0.047) levels, and 3) higher areas under the curve (AUC) for glucose (AUC-Glu, P = 0.025) and homeostatic model assessment for insulin resistance (HOMA-IR, P = 0.008). After treatment, compared with vehicle-treated PNA rats, cryptotanshinone-treated PNA rats had 1) improved estrous cycles (P = 0.045), 2) reduced 17-hydroxyprogesterone (P = 0.041), androstenedione (P = 0.038), testosterone (P = 0.003), glucose (P = 0.036), and insulin (P = 0.041) levels, and 3) lower AUC-Glu (P = 0.045) and HOMA-IR (P = 0.024). Western blot showed that cryptotanshinone reversed the altered protein expressions of insulin receptor substrate-1 and -2, phosphatidylinositol 3-kinase p85α, glucose transporter-4, ERK-1, and 17α-hydroxylase within PNA ovaries. We conclude that PNA model rats exhibit reproductive and metabolic phenotypes of human PCOS and that regulation of key molecules in insulin signaling and androgen synthesis within PNA ovaries may explain cryptotanshinone's therapeutic effects.

17 alpha-hydroxyprogesterone caproate vehicle, castor oil, enhances the contractile effect of oxytocin in human myometrium in pregnancy.

OBJECTIVE: The possibility exists that the vehicle for 17-alpha-hydroxyprogesterone caproate, castor oil, exerts an effect on human uterine contractility. The aim of this study was to evaluate its effects on contractility of myometrial preparations that were obtained during pregnancy. STUDY DESIGN: Myometrial strips were suspended under isometric conditions. Contractility was induced with oxytocin. Strips were incubated in castor oil or physiologic salt solution and suspended for a further oxytocin challenge. Contractile integrals were compared between both groups. RESULTS: Strips that were exposed to castor oil demonstrated increased contractile activity that was elicited by oxytocin (mean contractility value, 165.53%+/-17.03%; n=8; P=.004), compared with control strips (mean contractility value, 72.57%+/-7.48%; n=8; P=.003). There was a significant increase in contractile activity of the castor oil-exposed strips, compared with those that were exposed to physiologic salt solution (n=8; P<.001). CONCLUSION: Exposure of human myometrial preparations to castor oil results in enhanced oxytocin-induced contractility.

Pharmacologic actions of progestins to inhibit cervical ripening and prevent delivery depend on their properties, the route of administration, and the vehicle.

OBJECTIVE: The purpose of this study was to evaluate cervical changes and delivery at term during pregnancy in rats after various progestin treatments. STUDY DESIGN: Pregnant rats were treated by various routes and vehicles with progesterone, 17-alpha-hydroxyprogesterone caproate (17P), R5020, and RU-486. Delivery time was determined and cervical ripening was assessed in vivo by collagen light-induced fluorescence. RESULTS: The cervix is rigid in the progesterone injection, 17P, and vaginal R5020 groups vs controls. Vaginal progesterone had no effect. RU-486 treatment softened the cervix during preterm delivery. Only subcutaneous injected progesterone, R5020 (subcutaneous and vaginal), and topical progesterone in sesame and fish oil inhibits delivery. Delivery is not changed by subcutaneous injection of 17P, vaginal progesterone, oral progesterone, and topical progesterone in Replens (Crinone; Columbia Labs, Livingston, NJ). CONCLUSION: Inhibition of cervical ripening and delivery by progestins depends on many factors that include their properties, the route of administration, and the vehicle. This study suggests reasons that the present treatments for preterm labor are not efficacious.

[Controlling effect of berberine on in vitro synthesis and metabolism of steroid hormones in insulin resistant ovary].

OBJECTIVE: To investigate the functional and metabolic alterations in cultured insulin resistant ovary model in vitro, and to observe the effect of berberine (Ber, a Chinese medical monomer) in improving insulin resistance (IR). METHODS: Ovary of mouse was cultured in vitro and treated by dexamethasone (Dex) to induce IR for establishing IR model ovary. The functional alteration in model ovary was assessed through detecting glucose and hormone levels in medium using RT-PCR, meanwhile, the expression of key molecules in insulin signal and steroid synthetic pathway were detected, and condition of IR improved by berberine was evaluated also. RESULTS: (1) The model ovary was made by Dex in dose- and acting time-dependent manner. After being treated by 300 nmol/L Dex for 48 h, the glucose uptake of ovary reduced from 9.05 +/- 0.75 mg/g to 2.48 +/- 0.29 mg/g (P < 0.05); it further decreased (from 9.59 +/- 1.74 mg/g to 1.94 +/- 0.19 mg/g, P < 0.01) under the stimulation of insulin, which proved that the IR model ovary was made successfully. Berberine significantly increased the glucose uptake of model ovaries (1.89 +/- 0.33 mg/g to 13.95 +/- 3.30 mg/g, P < 0.05). (2) As compared with control group, levels of testosterone (T) and androstenedione (A2) were higher, and levels of progesterone (P) and 17-hydroxyprogesterone (17-OHP) were lower significantly in the model. Berberine reversed the alternations of T, A2 and 17-OHP levels, but did not influence the level of P. (3) RT-PCR showed that the mRNA expressions of cytochrome 17-hydroxylase (CYP17) and mini-chromosome maintenance protein-2 (MCM-2) elevated, but extracellular regulated protein-1 (ERK-1), protein kinase B (AKT-2) and glycogen synthase kinase-3 (GSK-3beta) lowered in the medium after Dex inducing. Berberine treatment restored these molecular index obviously. CONCLUSIONS: (1) Dex could induce IR in mouse ovary, which might enhance the androgenic synthesis. (2) Berberine could alleviate the degree of IR and the androgen synthesis, indicating that the Chinese sensitizing agents has favorable therapeutic effect for the treatment of polycystic ovaries.

Testosterone synthesized in cultured human SZ95 sebocytes derives mainly from dehydroepiandrosterone.

Human sebaceous gland possesses all the steroidogenic enzymes required for androgen synthesis. It remains unclear whether the testosterone produced in situ mainly derives from circulating dehydroepiandrosterone (DHEA) or from de novo synthesis utilizing serum cholesterol. Using testosterone radioimmunoassay, we found that testosterone was barely detectable in the supernatant of cultured human SZ95 sebocytes when cholesterol was added alone, indicating a low basal expression of steroidogenic acute regulatory protein (StAR) in SZ95 cells. Human chorionic gonadotropin and fibroblast growth factor-9 were as potent as forskolin in activating StAR to enhance testosterone production, while interleukin-1 beta, dexamethasone, insulin and insulin-like growth factor-1 showed no stimulatory effect. A two-fold increase of testosterone production was observed in supplementation of DHEA as compared to pregnenolone, progesterone or 17 alpha-hydroxyprogesterone. Based on our findings, testosterone synthesized in cultured sebocytes derived mainly from DHEA and inhibition of 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase may be a new target of androgen suppression for acne treatment.

Structure-activity relationships of a series of analogs of the endozepine octadecaneuropeptide (ODN(11)(-)(18)) on neurosteroid biosynthesis by hypothalamic explants.

We have previously shown that the endozepine octadecaneuropeptide (ODN) stimulates the biosynthesis of neurosteroids from frog hypothalamic explants. In the present study, we have investigated the structure-activity relationships of a series of analogs of the C-terminal octapeptide of ODN (OP) on neurosteroid formation. We found that OP and its cyclic analog cyclo1-8OP stimulate in a concentration-dependent manner the synthesis of various steroids including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and dehydroepiandrosterone. Deletion or Ala-substitution of the Arg1 or Pro2 residues of OP did not affect the activity of the peptide. In contrast, deletion or replacement of any of the amino acids of the C-terminal hexapeptide fragment totally abolished the effect of OP on neurosteroid biosynthesis. The present study indicates that the C-terminal hexapeptide of ODN/OP is the minimal sequence retaining full biological activity on steroid-producing neurons.

Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.

Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

Regulatory mechanism of Toona sinensis on mouse leydig cell steroidogenesis.

Toona sinensis (TS), a kind of arbor, widely distributes nowadays in Asia. The leaves of TS have been used as an effective nutritious food in Chinese society for a long time. It was reported that Toona sinensis can induce apoptosis of cancer cells, reduce plasma glucose in diabetic rats, and improve lipolysis of differentiated 3T3-L1 adipocyte and its uptake of glucose. It has also been shown that TS may increase dynamic activity of human sperm. Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity. Primary mouse Leydig cells were purified to conduct the in vitro experiments. Different concentrations of crude Toona sinensis were added to primary mouse Leydig cells and the testosterone production was determined. The results showed that crude TS significantly inhibited both basal and human chorionic gonadotropin (hCG)-stimulated testosterone productions in dose dependent manner, respectively (P<0.05). Crude TS also reduced the forskolin- and dibutyryl-cAMP (dbcAMP)-stimulated testosterone production (P<0.05), which indicated that crude TS might affect protein kinase A (PKA) signal transduction pathway at the site after the formation of cyclic AMP. Moreover, TS inhibited Leydig cell steroidogenesis by suppressing the activity of steroidogenic enzymes including P450 side chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 20 alpha-hydroxylase and 17 beta-hydroxysteroid dehydrogenase (P<0.05). In summary, these results suggested that TS inhibited steroidogenesis by suppressing the cAMP-PKA signaling pathway and the activities of steroidogenic enzymes in normal mouse Leydig cells.

Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements.

Direct measurement of the fluorescence lifetime (FLT) of a fluorescent label is an emerging method for high-throughput screening. Changes in the fluorescence lifetime can be correlated to changes in the non-radiative relaxation pathway(s) for the excited state of the label. These pathways can be environmentally sensitive, such as when a labeled analyte is free in solution versus bound to a receptor. Because lifetime is an intrinsic property of a fluorophore, it is not concentration dependent, and therefore has advantages similar to those of ratiometric fluorescent techniques such as fluorescence resonance energy transfer or fluorescence polarization. We have applied the FLT measurement technique to a screen of a small compound library in order to identify compounds that bind to the progesterone receptor, and compared the results to those obtained by performing the assay in fluorescence polarization mode. Each readout modality showed excellent Z'; values, with the FLT readout performing slightly better in this respect. Interfering compounds could be rapidly identified for either assay format by comparing the results between the two formats.

Ovarian steroids modulate neuroendocrine dysfunction in polycystic ovary syndrome.

OBJECTIVE: Neuroendocrine dysfunction in polycystic ovary syndrome (PCOS) was addressed by studying the steroid hormone changes in women with PCOS with either high or normal LH levels leading to inferences regarding the primacy of elevated LH in the pathophysiology of PCOS. METHODS: A cross-sectional study was designed in an academic clinical facility involving 234 women with PCOS. Patients were divided into two groups based on an LH/FSH ratio < or >1 and hormonal and metabolic studies were performed in both groups. Factors were determined by binomial logistic regression that predicted group membership of these women. RESULTS: Higher follicular phase estradiol (E2) and androstenedione (A4) levels as well as greater insulin sensitivity were the only factors that predicted the presence of neuroendocrine dysfunction with elevated A4 being necessary for neuroendocrine dysfunction. CONCLUSIONS: It was concluded that uncoupling of hypothalamic E2 inhibition by elevated ovarian A4 associated with E2 related sensitization of pituitary LH leads to neuroendocrine dysfunction in PCOS.

Molecular cloning and expression of guinea pig cytochrome P450c21 cDNA (steroid 21-hydroxylase) isolated from the adrenals.

In mammals, the P450c21 enzyme mediates 21-hydroxylase activity by transforming progesterone and 17-hydroxyprogesterone into deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC), respectively. Previous studies have shown that among the adrenal steroid hydroxylase enzymes involved in C19 steroid and glucocorticoid syntheses, P450c21 plays an important role, because it is localized at the key branch between glucocorticoids and C19 steroid production. Its implication in congenital adrenal hyperplasia is also of great clinical interest. In this study, in addition to describing the isolation of the P450c21 cDNA from guinea pig (GP) adrenal and comparing it to those from other species, we report on its tissue-distribution and on the activity of the recombinant protein towards progesterone and 17-hydroxyprogesterone. The guinea pig P450c21 includes the full-length coding region (1464 nucleotide) that is translated to a protein of 488 amino acids. The clone shares highly conserved regions with other species. The guinea pig P450c21 cDNA hybridized with a major transcript of 2.1kb and with two minor related transcripts of 1.8 and 1.5 kb and was found to be adrenal-specific among the various tissues analyzed. Characterization of the enzymatic activity by transient transfection of the guinea pig P450c21 cDNA in human embryonic kidney 293 cells indicated a net preference for the 21-hydroxylation of 17-hydroxyprogesterone in comparison to the progesterone substrate. Assays showed a maximum conversion rate of 12.5% for the conversion of progesterone into deoxycorticosterone (mineralocorticoid pathway), whereas the guinea pig P450c21 demonstrated a higher activity with 17alpha-hydroxyprogesterone, with 55% of 11-deoxycortisol formation (glucocorticoid pathway) after 48 h. Adrenocorticotropin and an analogue of the second messenger cyclic adenosine monophosphate specifically increased the abundance of P450c21 mRNA levels in guinea pig adrenal cells.